An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

• Published in: Nature Vol. 432; no. 7020; pp. 1036 - 1040
• Main Authors: Buchholz, Frank, Kittler, Ralf, Putz, Gabriele, Pelletier, Laurence, Poser, Ina, Heninger, Anne-Kristin, Drechsel, David, Fischer, Steffi, Konstantinova, Irena, Habermann, Bianca, Grabner, Hannes, Yaspo, Marie-Laure, Himmelbauer, Heinz, Korn, Bernd, Neugebauer, Karla, Pisabarro, Maria Teresa
• Format: Journal Article
• Language: English
• Published: London Nature Publishing 23-12-2004; Nature Publishing Group
• Subjects: Biological and medical sciences; Cell cycle, cell proliferation; Cell division; Cell Division - genetics; Cell physiology; Cell Proliferation; Cell Survival; Cytokinesis - genetics; Endoribonucleases - metabolism; Fundamental and applied biological sciences. Psychology; Gene Library; Genes, Essential - genetics; Genomics; Genomics - methods; HeLa Cells; Humans; Interferon; Mammals; Microscopy, Video; Mitosis - genetics; Molecular and cellular biology; Molecular Sequence Data; Phenotype; Ribonucleic acid; RNA; RNA Interference; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Spindle Apparatus - physiology; Human; Cell division; Screening; RNA interference; Gene; Genome
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature03159

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.